Amended diagnosis, mitochondrial genome, and phylogenetic position of Sphyranura euryceae (Neodermata, Monogenea, Polystomatidae), a parasite of the Oklahoma salamander

Polystomatidae is a monogenean family whose representatives infect mainly (semi)-aquatic tetrapods. Species of Sphyranura Wright, 1879 exhibit ectoparasitism on salamander hosts, with molecular work supporting their inclusion within Polystomatidae, at an early diverging, yet unresolved, position in the clade of otherwise endoparasitic polystomatid parasites of batrachian hosts. Records of representatives of Sphyranura are scarce with genetic data only available for S. oligorchis Alvey, 1933. Based on detailed morphological examination and comparison with type material, we identified worms belonging to Sphyranura infecting Oklahoma salamander (Eurycea tynerensis) as S. euryceae Hughes & Moore, 1943. Along with an amended diagnosis of Sphyranura, we provide the first molecular data for S. euryceae in the form of a mitochondrial genome and nuclear (18S, 28S rRNA) markers. Close morphological similarity between the two species of Sphyranura is reflected in low genetic divergence. Mitochondrial level comparison reveals instances of tRNA gene rearrangements in polystomatids. Although the phylogenetic reconstruction supports Sphyranura as early branching in the lineage of polystomatid monogeneans infecting batrachians, certain nodes remain unresolved.

The polystomatid genus, Sphyranura Wright, 1879 is restricted to North America and its members infect the gills and skin of salamanders. Sphyranura consists of S. osleri Wright, 1879, S. oligorchis Alvey, 1933, S. polyorchis Alvey, 1936 and S. euryceae Hughes & Moore, 1943. It has been argued, however, that S. polyorchis cannot be justified as a separate species from S. osleri on the basis of minor morphological differences [41]. Sphyranura osleri, S. oligorchis and S. polyorchis parasitise the Common mudpuppy (Necturus maculosus Rafinesque), with records of S. oligorchis also parasitising the Red River waterdog (Necturus louisianensis Viosca) [51]. Sphyranura euryceae is a parasite of the Oklahoma salamander (Eurycea tynerensis Moore & Hughes) [23], a plethodontid salamander endemic to the Ozark Plateau. Adults of this species exhibit alternative life histories with paedomorphic populations associated with chert streambeds where they can access subsurface water year-round and metamorphic populations associated with compact streambeds where such access is not guaranteed [10,19]. More recently, S. euryceae has been observed in the Cave salamander (Eurycea lucifuga Rafinesque) [36] and Western Grotto salamander (Eurycea spelaea Stejneger) [37]. In general, there is a scarcity of records of representatives of Sphyranura and relatively little knowledge about the genus besides morphology and principal host distribution. However, given the intervening decades since Hughes & Moore's [23] description of S. euryceae, advances in staining procedures and microscopy allow for a more detailed morphological examination than was possible at the time of description. Thus, descriptions of representatives of Sphyranura often lack some of the morphological information available for more recently studied monogeneans.
Sphyranura was long assigned to Sphyranuridae [40], and considered a sister group to Polystomatidae on the basis that its members possess a single pair of haptoral suckers in contrast to three pairs found in other polystomatids [38]. Sinnappah et al. [46], however, inferred a phylogeny of Polystomatoinea based on partial sequences of the 18S rDNA marker, which confidently placed Sphyranura within Polystomatidae. These authors further proposed that the morphological differences between Sphyranura and Polystomatidae as described above are the result of an evolutionary retention of juvenile characters in adults within Sphyranura [46]. However, this phylogeny only included seven representatives of Polystomatidae and a single representative of Sphyranura. Furthermore, the position of Sphyranura within batrachian polystomes was not well supported. Subsequent work, also based on partial 18S rDNA sequences, split Polystomatidae into two lineages: one parasitising exclusively amphibians, the other parasitising mainly chelonians. This phylogeny also supported Sphyranura as being nested within the lineage of anuran polystomatids, its exact relationships, however, remained unresolved [53]. More recently, Héritier et al. [22] inferred the phylogeny of Polystomatidae based on the complete 18S rDNA sequence, a partial 28S rDNA sequence and two partial sequences of mitochondrial genes, cox1 and 12S rDNA, which supported the division of Polystomatidae into the "Polbatrach" and "Polchelon" (acronyms coined by the authors) lineages with Concinnocotyla australensis (Reichenbach-Klinke, 1966), a parasite of the Australian lungfish (Neoceratodus forsteri (Krefft)), branching off prior to this split. The former lineage includes all polystomatids of batrachian hosts (Caudata and Anura), whilst the latter includes all polystomatids of chelonian hosts as well as Nanopolystoma tinsleyi du Preez, Badets & Verneau, 2014 of the Cayenne caecilian (Typhlonectes compressicauda Duméril & Bibron) and Oculotrema hippopotami Stunkard, 1924 of the common hippopotamus (Hippopotamus amphibius L.). Furthermore, this phylogeny suggested that Sphyranura is an early, although unresolved, branching lineage within the "Polbatrach" polystomatids [22]. This phylogeny therefore supported the hypothesis of an origin of Polystomatidae prior to the colonisation of terrestrial environments by tetrapods followed by hostparasite coevolution as different tetrapod lineages diverged [55].
In the present study, we aim to produce an amended diagnosis of Sphyranura using various staining techniques to provide morphological characters at a higher resolution than previous work. Further, we provide the first molecular sequences for a member of Sphyranura other than S. oligorchis, including its mitogenome. Although beyond the scope of the current research, this mitogenome may provide a valuable resource in future phylogenetic studies of Monogenea. Given the unresolved position of Sphyranura, questions regarding the number of evolutionary colonisations of caudatan hosts by polystomatid monogeneans remain. We therefore present an updated phylogeny of Polystomatidae, including the new specimens and several other polystomatid taxa made available since the publication of that inferred by Héritier et al. [22] in 2015, including those submitted by Du Preez and Verneau [18] in 2020.
were collected with an aquatic dipnet at Greathouse Spring in Tontitown, Benton County, Arkansas, USA (Coordinates 36°8 0 11.1192 00 N, À94°12 0 10.0764 00 W). Specimens were placed in habitat water and examined for ectoparasites within 24 h. Salamanders were killed with an overdose of a concentrated solution of tricaine methanesulfonate and their gills and body were examined under a stereomicroscope. When monogeneans were observed on gills, they were removed and relaxed in hot tap water and stored in either 10% neutral-buffered formalin (NBF) or 98% molecular grade ethanol.

Staining procedure
Seven adult individuals and two larvae used for morphological analysis were selected from those preserved in 10% NBF. These were then stained with various media and mounted on standard microscope slides to be morphologically characterised. The staining procedure included the following steps: Individual worms were first placed in a solution of 70% ethanol to be dehydrated before being overstained using a 1:1 mixture of acetocarmine (or Schneider-acetocarmine in the case of specimens 4, 6 and larva 1) and 70% ethanol (>12 h). The ethanolacetocarmine mix was then gradually washed out using acid alcohol until internal structures such as testes, ovaries and vesicles were visible under a binocular microscope. At this point, the process was halted by washing in distilled water for 5 min to remove excess acetocarmine. Specimens 1 and 3 were then stained with Astra blue for 40 min before being washed twice in distilled water to wash out residual Astra blue [47]. This step was skipped for specimens 2, 4, 5, 6, 7 and the two larvae. After this, specimens were dehydrated through a series of increasing ethanol concentrations (5 min at 70%, 5 min at 80%, 15 min at 96%, 5 min at 100%) and carboxyl was added. Xylene was then added to clear the specimens and they were mounted on a slide using Canada balsam, ensuring that the specimens were lying flat when the cover slip was added. The slides were then weighted to ensure specimens remained flat and given two weeks on a radiator to dry out. The attachment structures of two individuals were placed on a slide in a drop of water that was subsequently replaced by Hoyer's medium and covered with a cover slip that was sealed with Glyceel [3].

Morphological characterisation
The morphological part of the study was done using Leica DM 2500 LED microscopes (Leica Microsystems, Wetzlar, Germany) and the software LasX v3.6.0 using Differential Interference Contrast (DIC) and Phase Contrast, where necessary, to gain optimal view of individual anatomical features. In total, 35 morphological characters including hard and soft parts were measured following the terminology of [43]. A comparison of the new specimens with existing type material belonging to Sphyranura provided by the American Museum of Natural History (AMNH) was undertaken to further support the species identification of these specimens with re-measurements of type material being undertaken where necessary and possible. The material included two specimens of S. osleri (accession numbers AMNH 1427.1 and AMNH 1427.2), one specimen of S. polyorchis (accession number

DNA extraction and PCR
Genomic DNA was extracted from four individuals using a Quick-DNA TM Miniprep Plus Kit (Zymo Research Irvine, CA, USA), following the manufacturer's instructions with minor modifications, specifically: initial incubation overnight, and elution in 2 Â 50 lL after 10 min incubation at room temperature, each. DNA was then quantified with a Qubit fluorometer (dsDNA HS assay). The DNA concentration of the individual extracts measured between 0.665 and 1.34 ng/lL. The partial 12S, 28S and 18S rRNA genes of four specimens were then amplified and sequenced. Primers used for amplification and sequencing of each gene were selected based on previous work [22,54] and were as follows: 18S: IR5/L7, 12S: 12SpolF1/ 12SpolR9, for the 28S two overlapping fragments of unequal length were sequenced. LSU5/IR14 primers were used for larger of these and IF15/LSU3 for the smaller. The reactions were performed in a total volume of 11.2 lL, including 7.05 lL water, 1.0 lL buffer (BioTherm 10Â PCR Buffer, 15 mM MgCl 2 ), 0.35 lL dNTPs (10 mM), 0.25 lL each of forward and reverse primers (0.1 mM), 0.3 lL Taq polymerase (SupraTherm 5 units/lL) and 2.0 lL DNA template. The amplification cycle consisted of a step of 3 min at 95°C for initial denaturation; 45 cycles of 30 s at 95°C for denaturation, 30 s at 50°C for annealing and 1 min at 72°C for elongation; one final step of 7 min at 72°C for terminal elongation. The PCR products were visualised on agarose gels in order to verify the success of PCR amplification before sequencing. The PCR products were purified by adding a mixture of 0.5 lL ExoSAP (ExoSAP-IT: Amersham Biosciences) and 1.2 lL water to each and incubating in a thermocycler for 45 min at 37°C, followed by 15 min at 80°C. The sequencing reaction was run using a cycle beginning with a single step of initial denaturation for 3 min at 94°C; 35 cycles of 30 s at 94°C, 30 s at 50°C, and 3 min at 60°C; one final step of 7 min at 60°C. Sequencing products were purified with SephadexTM G-50 (GE Healthcare Chicago, IL, USA) and sequenced on an ABI 3130xl capillary sequencer (Applied Biosystems, Waltham, MA, USA). All newly generated sequences have been deposited on GenBank (see Table 1).

Mitogenome assembly and annotation
DNA extracts of two specimens (SPY1 and SPY2) were sent for whole genome sequencing to commercial sequencing centres. For SPY1, library preparation (Nextera XT, 550 bp insert size) was performed by Macrogen Inc. (Seoul, Korea). For SPY2, library preparation (NEBNext Ò Ultra IIDNA Library Prep Kit, 550 bp insert size) was done by Novogene (Cambridge, UK). Libraries were sequenced on NovaSeq 6000 systems (2 Â 150 bp) at the respective centres. Raw read data were first trimmed using Trimmomatic v.0.38 [9] and the following parameters: a minimum length of 40 bp, a window size of 5 and required quality per window of 15 and a leading and trailing quality of 3. For both specimens, a subsample of 10 000 000 trimmed reads was randomly selected using seqtk v.1.3 [45] with the seed 553353 and fed into the assembly process. A successful assembly of SPY2 was retrieved using GetOrganelle v. 1.7.1 [24]. The first and last 200 bp of this result were joined and trimmed reads were mapped back to this fragment using MITObim v.1.9.1 [21]. Reads mapping full length without any conflict across this tentative junction were taken as verification of circularity. A full-length mitochondrial genome of SPY1 could not be recovered using GetOrganelle, so this sample was assembled via MITObim, using the successful SPY2 assembly as a reference. For this result, circularity was confirmed using the script circules.py shipping with MITObim. Annotation was then performed via MITOS v.1.0.5 [7] using the genetic code 09 (Echinoderm/Flatworm Mitochondrial). Upon initial visual inspection and comparison of protein-coding genes with those of other monogeneans, it became apparent that there were errors in the start and end positions of many protein coding genes given by MITOS v.1.0.5. The assembly was subsequently submitted to MITOS2 via webserver [6]. In addition to MITOS v.1.0.5, the coordinates and secondary structure of mitochondrial tRNA genes were confirmed using ARWEN v.1.2 [32]. In cases where the coordinates given by MITOS v.1.0.5 did not match those of ARWEN v.1.2, those provided by ARWEN v.1.2 were used, provided a 6-7 bp acceptor stem was present. The cox1 and 12S sequences for the samples SPY1 and SPY2 were retrieved from the mitochondrial genomes based on the annotation results from MITOS2. The mitochondrial genome of SPY1 was compared with that of Diplorchis hangzhouensis (Accession: JQ038227.1), the only polystomatid species of which the mitochondrial genome is available. Two mitochondrial genomes of S. euryceae (SPY1 and SPY2) were deposited on NCBI GenBank under the accession numbers OP920606 and OP920607.

Extracting full length 18S and 28S
Whilst only partial 18S sequences were retrieved via Sanger sequencing, the complete 18S sequence could be extracted from  Table 1. List of parasite taxa and their respective host species, country of origin and GenBank accession numbers of the markers used to infer the phylogeny. Taxa marked with * were not included in the phylogeny of Héritier et al. [22]. Taxa marked with ** were renamed since the publication of Héritier et al. [22] [14], with original names in brackets. In these cases, the GenBank accession numbers correspond to original names.

Species
Host  WGS data for the samples SPY1 and SPY2. This was done first using MITObim v.1.9.1 using the 18S sequence retrieved from Sanger sequencing as an initial seed to extend from the readpool of WGS data, interleaved using BBmap v.38.90 [11]. Barrnap (BAsic Rapid Ribosomal RNA Predictor) v.0.9 [44] was then employed to predict the location of the 18S sequence within the assembled data. The same method was employed to retrieve the full 28S sequence, with the partial 28S sequence, produced via Sanger sequencing used as the initial seed. Due to the low coverage of SPY1, an initial assembly could not be retrieved from WGS data using the partial 18S and 28S sequences as seeds. Instead, the assembled sequences of SPY2 were used as references for assembly via MITObim.
Barrnap was subsequently run on the completed SPY1 assemblies to infer the positions of 18S and 28S, respectively.

Phylogenetic analysis
In addition to sequences obtained from the new specimens, sequences representing a further 66 polystomatid taxa and three non-polystomatid monogeneans were accessed via NCBI GenBank. Taxa included in this phylogenetic analysis were selected based on the availability of sequences on NCBI GenBank. A given taxon was included in the analysis on the basis that at least two of the four markers (12S, 18S, 28S and cox1) were present. Partial sequences were included provided they overlap at least in part with the sequences of all other taxa for which sequence data of a given marker was included. In addition to the 55 polystomatid taxa presented in the analysis of Héritier et al. [22], sequences from a further 15 polystomatids were included in addition to the new specimens of Sphyranura. Species of Gastrocotylidae (Pseudaxine trachuri Parona & Perugia, 1890), Diclidophoridae (Neoheterobothrium hirame Ogawa, 1999), and Microcotylidae (Microcotyle sp.) were selected as an outgroup in line with Héritier et al. [22]. Accession numbers of these sequences as well as information on the respective host species, country of origin and site of infection are provided in Table 1.
A maximum likelihood phylogeny was inferred from a subset of the total taxa, representing the clade of polystomatid parasites of batrachian hosts, referred to as the "Polbatrach" clade by Héritier et al. [22]. The list of taxa used in this phylogeny is shown in Table 1. Sequences representing these taxa, as well as an outgroup comprising C. australensis, were aligned using MAFFT T v.7.464 [26] and trimmed using TrimAl v.1.4.1 [13] in "strict" mode. The four separate alignments were then concatenated into a single alignment using the script concat.py v.0.21 (https://github.com/reslp/concat). PartitionFinder2 [30] selected a GTR+I+G model for the 12S and 18S sequences, a TVM+I+G model for the 28S sequence, and TRN+I+G, TIM+I+G, and GTR+I+G, respectively for the three codon positions of cox1. Additionally, phylogenies representing the entire taxa set were inferred via two methods. In the first, the four sequence sets were aligned per marker using MAFFT and trimmed using TrimAl "strict mode". Alignments were inspected visually in AliView v.1.28 [31]. Sequences were concatenated into a single alignment as above. For the second method we performed RNA specific alignment using predicted secondary structure for 18S and 28S rRNA markers using R-COFFEE [56], as implemented in T-COFFEE v.11.00 [50]. Since this algorithm does not accept ambiguous nucleotides, we removed any sequences that contained more than one ambiguity characters. For sequences with a single ambiguity character only, the ambiguous character was replaced randomly with one of the candidate characters (custom script replace_ IUPAC.py) prior to alignment with R-COFFEE, and the original ambiguity was restored after alignment (custom script restore.py). Alignments were subsequently trimmed as above using TrimAl. The best fitting partitioning schemes for the three ribosomal sequences as well as the three codon positions of the cox1 gene were selected by PartitionFinder2 using the "greedy search" algorithm. PartitionFinder2 selected a GTR +I+G model for all subsets in the MAFFT alignment, and GTR +I+G for the 12S and 18S sequences as well as the three codon positions of cox1 and the GTR+G model for 28S in the R-COFFEE alignment.  [39] was then used to infer a Maximum Likelihood phylogeny of all three alignments. Phylogenetic trees were visualised using the web-based tool ITOL (Interactive Tree Of Life) [34].

Taxonomic account
Family Polystomatidae Gamble, 1896 Genus Sphyranura Poche, 1925 Amended diagnosis of Sphyranura Poche, 1925 Body elongated with greatest body width found approximately half to two-thirds of distance between haptor and the oral sucker. Body width (measured at widest point) 17-45% of body length with variation between both species and individuals (Table 2). Oral suckers either terminal or subterminal varying in width from 105-300 lm. Single pair of roughly circular haptoral suckers and of anchors, seven pairs of marginal and one pair of acetabular hooks situated at basal end of body. Interior haptoral sucker width accounts for 61-68% of haptor width. Haptor length accounts for 14-19% of body length and haptor width accounts for 26-110% of body width. Vitellaria arranged laterally on both sides of the body extending from region of uterus to peduncle, accounting approximately for two thirds of body length.   [23,36] including re-measurement of type material of S. osleri, S. oligorchis and S. polyorchis [1]. Range is followed by the mean in parentheses. Infection site: Skin mainly at the base of legs, and external gills.
Infection parameters: Current studyin 2019, 12 specimens of E. tynerensis out of 27 infected (prevalence = 44.4%) with one or two individuals per host; in 2020, two out of six specimens of E. tynerensis infected (prevalence = 33.3%) with one individual. McAllister [36] reported infection in ten out of ten specimens of E. lucifuga, and ten out of ten specimens of E. tynerensis (prevalence = 100%).
Morphological characters. Small fusiform worms with a subterminal oral sucker at one end of the body and a single pair of haptors at the other. The oral sucker is followed by the pharynx which is wide and oval tapering to a narrow point at the anterior end. With the exception of the haptors, the body's widest point is situated roughly two thirds of the way along the body starting from the peduncle. From the peduncle to this widest point of the body is situated a mass of vitellaria. Testes were observed in four of the seven adult specimens, numbering between 5-7 per individual and were arranged in a single line along the centre of the body and were in some cases at least partially obscured by the vitellaria. The ovary was observed in all adult specimens in the study, situated anterior to the testes and vitellaria. Intra-uterine eggs were observed in two specimens. A spherical genital bulb with conical spines is situated anterior to the ovary and connected to the testes via the vas deferens, although this latter was only observable in one specimen. Two roughly circular haptoral suckers were situated laterally to the posterior end of the body. Each haptor possessed several marginal hooklets in addition to a much larger anchor which exhibits an accessory sclerite at the base of a larger recurved hook and a deep, triangular cut between the inner and outer roots. Measurements of the aforementioned features, both on new specimens and type material, as well as previous data on Sphyranura spp. are presented in Table 2. In addition to the seven adult specimens, morphological characteristics of two larvae were taken. Micrographs showing morphological features of S. euryceae are presented in Figure 2.
Differential diagnosis. Sphyranura euryceae may be distinguished from congeners on a number of morphological features. First, the overall body shape is more elongated than that of congeners (body width as a proportion of body length = 20% vs S. osleri = 36%, S. polyorchis = 31% and S. oligorchis = 28%), although there is some degree of overlap with S. oligorchis, but not with S. osleri and S. polyorchis. Further, haptor width as a proportion of body width is much greater in S. euryceae compared to the others (S. euryceae = 65% vs S. osleri = 34%, S. polyorchis = 40% and S. oligorchis = 51%). The oral sucker of S. euryceae is sub-terminal rather than terminal as in the other members of the genus. The mean anchor length of S. euryceae is also less than that of congeners although there is overlap between all species in this trait.

Mitochondrial genome
Mitochondrial genomes were assembled for the samples SPY1 and SPY2, a representation of which is presented in Figure 3. The assembly of SPY2 was performed using GetOrganelle from a subsample of 10 million reads, 41 406 of which were used post-filtering to assemble the mitochondrial genome. The assembly had a total length of 13 728 bp and an average coverage of 201. Annotation of this assembly reveals the presence of 12 protein coding genes (the absence of atp8 is a characteristic of Neodermata [48]). Three non-coding regions with elevated AT content were found between cox1 and rrnL (469 bp, 78% AT), nad6 and nad5 genes (738 bp, 79% AT) and cox2 and cox3 genes (439 bp, 74% AT). De novo assembly of SPY1 was attempted using both GetOrganelle and MITObim but did not successfully produce a full-length mitochondrial genome. However, when assembled using MITObim using the assembly of SPY2 as a reference, a full mitochondrial genome was recovered from a subsample of 10 million reads, 12 310 of which were mitochondrial. The two sequences were nearly identical with the following exceptions shown in Table 3. In addition to these differences, there was a region of high dissimilarity between the positions 5545 and 5996.
This dissimilarity was likely due to the presence of AT repeats which rendered this region difficult to assemble. Coverage differed between the two samples and is indicated in Table 4. A comparison of this mitochondrial genome with that of D. hangzhouensis is provided in Table 5. Overall, the two tRNA-genes missing in the original annotation of D. hangzhouensis, trnV and trnA, were found (see Table 5). Gene order differences of adjacent features between the two polystomatid species include the following two cases. In S. euryceae, trnS2 precedes trnL2 whereas in D. hangzhouensis, this is reversed. In S. euryceae, we see the sequence trnK/nad6/trnY whereas in D. hangzhouensis, we see trnY/nad6/trnK.

Phylogeny
Sequences of S. euryceae were highly similar to those of S. oligorchis with percentage identities of 93.6% for 12S (481 bp), 99.4-99.5% for 18S (2009 bp), 100% for 28S (1411 bp) and 96.9-97.4% for cox1 (395 bp). Intraspecific variation within S. euryceae reaches 0.002% in the portion of cox1 region. A Maximum Likelihood tree was inferred from a restricted taxa set representing the 42 polystomatids that make up the 'Polbatrach' clade and were aligned using MAFFT (Figure 4). A further two Maximum Likelihood trees were inferred from 77 taxa (including 74 polystomatids and three non-polystomatid monogeneans) based on alignments produced in MAFFT ( Figure 5) and R-COFFEE ( Figure 6) and in all trees, specimens of S. euryceae formed a monophyletic group that formed a sister-group relationship with S. oligorchis at an early branching, but unresolved position within the clade dubbed 'Polbatrach' by Héritier et al. [22]. However, the three trees present conflicting topologies and are characterised by low support values, making it impossible to determine the true evolutionary relationship of Sphyranura to other polystomatid parasites of batrachian hosts.

Status of Sphyranura euryceae
We provide an amended diagnosis of Sphyranura and obtained the first-ever molecular sequence data for S. euryceae. The divergence between S. euryceae and S. oligorchis appears Table 3. Positions of mismatches between the sequences of SPY1 and SPY2 and the gene in which these are found.
Position (s)  946  1021  4812-4814  7235  8115  12 040  12 513  12 918  SPY1  A  A  TAA  T  G  G  C  G  SPY2  T  T  -C  A  A  T  T  Gene  cox1  cox1  cox3  nad5  cob  trnA  nad1  nad1   Table 4. Library preparation kits and mitochondrial coverage of the sequences of SPY1 and SPY2. rather low compared to other congener polystomatid species. Species of Metapolystoma for instance exhibited 7.1-14.9% divergence in cox1 [29]. However, given that the two species are found on different hosts with non-overlapping ranges as well as the observed morphological differences, we argue that these represent two species, as traditionally described. The high molecular similarity of these sequences indicates that the split between these species was indeed recent. Comparison at the mitochondrial genome level revealed instances of gene order differences in polystomatids. Sphyranura was long thought to belong to Sphyranuridae. This was contradicted by the first molecular phylogenies, which placed it at an early-diverging, yet currently unresolved, position in the clade of polystomatids infecting batrachian hosts [22,53]. The inclusion of a second species of Sphyranura as well as 15 polystomatid taxa not included in the phylogeny by Héritier et al. [22] indicates an early branching Sphyranura within this clade. However, as in previous phylogenies [22,53] support for this position was ambiguous.

ID
Morphological comparison of Sphyranura spp.
Morphological analysis of the new specimens of S. euryceae and comparison of these with type material of S. osleri, S. oligorchis and S. polyorchis revealed high levels of both variability between conspecific individuals and overlap between each of the four species. It is important to note that individuals measured in this study as well as previous studies may well represent different life stages and may well have experienced different conditions prior to collection. Furthermore, the body tissues of monogeneans, with the exception of the sclerotised attachment organs, are soft and may not lie Table 5. Comparison of mitochondrial genomes of Sphyranura euryceae (SPY2 -NCBI GenBank accession number OP920606) and Diplorchis hangzhouensis (NCBI GenBank accession number JQ038227.1) including start and end positions of each feature, the start and stop codons of protein-coding genes and anticodons of tRNA genes. Positions given for D. hangzhouensis are as provided on NCBI. However, the trnA and trnV genes were not included on the NCBI annotation but were found in the present study, when reannotating the D. hangzhouensis genome with MITOS2 (indicated with *) or Arwen (**). completely flat during slide preparation. For these reasons, relative measurements should be used rather than absolute measurements for species differentiation. That said, the following features provided an informative diagnosis of S. euryceae: an overall body shape which was elongated compared to congeners; greater haptoral sucker width in relation to body width; and a sub-terminal, rather than terminal oral sucker. Finally, anchor length of S. euryceae was also less than that of congeners. It should also be noted that type material measured in this study represented only a single individual of S. polyorchis, of which many features were impossible to observe and measure. Sphyranura osleri was represented by two individuals, both deposited in 1879 and perhaps due to their age, many features were again impossible to measure. Based on this, no definite conclusion should be drawn regarding the validity of S. polyorchis as questioned by Price [42].   Mitochondrial genome of Sphyranura euryceae

Sphyranura euryceae
We provide the first available mitochondrial genome for Sphyranura and the second only for Polystomatidae. This mitochondrial genome may provide particular value for future phylogenetic work due to the fact that currently available mitogenomes for the sub-class Polyopisthocotylea are all, with the exception of D. hangzhouensis, from the order Mazocraeidea [2]. Furthermore, a second polystomatid mitogenome allows for the first insights on the gene order rearrangements in Polystomatidae. As with the majority of flatworm mitochondrial genomes available so far, 12 protein coding genes were found, with atp8 being absent [48]. A further 22 tRNA genes and the genes coding for both the large and small subunits of the mitochondrial rRNA were present. Comparison with the mitochondrial genome of D. hangzhouensis reveals similar gene order, with two instances of rearrangement in the order of adjacent tRNA genes between the two species. However, the order of protein coding genes was conserved between the two species. This is consistent with observations in other monogenean families such as Dactylogyridae [12,27] and Capsalidae [57], which exhibit rearrangements in the order of tRNA genes between species but generally not in protein coding genes. However, this should not be taken at face value as gene order in some groups of flatworms has been shown in some instances to be highly variable. Rearrangements in protein coding gene order have, for example, been observed within the genus Schistosoma [33]. Whether, and to what extent, such rearrangements exist in Polystomatidae therefore warrants further study as additional mitochondrial genomes become available. We identify differences in start/stop codon usage in eight of 12 protein coding genes between the two polystomatids. Furthermore, the abbreviated stop codon (TA-) was used in cox1 of S. euryceae, whereas this stop codon was TAA in D. hangzhouensis. The fact that the mitochondrial genome of SPY1 could not be assembled de novo indicates that when performing library preparation with low input data, the NEBNext Ò Ultra IIDNA Library Prep Kit is preferable to Nextera XT.

Phylogenetic position of Sphyranura
As first suggested by Sinnappah et al. [46] and supported by Héritier et al. [22], our phylogeny places Sphyranura within the 'Polbatrach' clade of Polystomatidae, rendering Sphyranuridae invalid. Although not fully supported, our phylogeny indicates Sphyranura to be an early branching lineage of the 'Polbatrach' clade. Moreover, two independent transitions to caudatan hosts are suggested, though low support of the early branching lineages restricts us from drawing final conclusions. Sphyranura oligorchis and S. euryceae formed a monophyletic group with little distance between them. Given this phylogenetic proximity and the overlap of many morphological characters seen here, it seems likely that the divergence of the two species occurred in the relatively recent past, following the acquisition of alternative host species by the ancestor of S. euryceae.
Apart from members of Sphyranura, Pseudopolystoma dendriticum Osaki, 1948, also parasitises the Japanese clawed salamander, Onychodactylus japonicus (Houttuyn). The two species are not closely related, thus indicating two independent acquisitions of urodelan hosts. Unlike the hosts of Sphyranura, O. japonicus goes through a full metamorphosis, during which larvae lose their external gills [52]. As a result, the acquisition of caudatan hosts by the ancestor of P. dendriticum was accompanied neither by a shift to ectoparasitism nor a retention of larval morphology as seen in Sphyranura.
Apart from members of Sphyranura, Pseudopolystoma dendriticum Osaki, 1948, also parasitises the Japanese clawed salamander, Onychodactylus japonicus (Houttuyn). The two species are not closely related, thus indicating two independent acquisitions of urodelan hosts. Unlike the hosts of Sphyranura, O. japonicus goes through a full metamorphosis, during which larvae lose their external gills [52]. As a result, the acquisition of caudatan hosts by the ancestor of P. dendriticum was accompanied neither by a shift to ectoparasitism nor a retention of larval morphology as seen in Sphyranura.